Response Surface Methodology (RSM)-Based Optimization of Ultrasound-Assisted Extraction of Sennoside A, Sennoside B, Aloe-Emodin, Emodin, and Chrysophanol from Senna alexandrina (Aerial Parts): HPLC-UV and Antioxidant Analysis

In this study, ultrasound-assisted extraction conditions were optimized to maximize the yields of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol from S. alexandrina (aerial parts). The three UAE factors, extraction temperature (S1), extraction time (S2), and liquid to solid ratio (S3), were optimized using response surface methodology (RSM). A Box–Behnken design was used for experimental design and phytoconstituent analysis was performed using high-performance liquid chromatography-UV. The optimal extraction conditions were found to be a 64.2 °C extraction temperature, 52.1 min extraction time, and 25.2 mL/g liquid to solid ratio. The experimental values of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol (2.237, 12.792, 2.457, 0.261, and 1.529%, respectively) agreed with those predicted (2.152, 12.031, 2.331, 0.214, and 1.411%, respectively) by RSM models, thus demonstrating the appropriateness of the model used and the accomplishment of RSM in optimizing the extraction conditions. Excellent antioxidant properties were exhibited by S. alexandrina methanol extract obtained using the optimized extraction conditions with a DPPH assay (IC50 = 59.7 ± 1.93, µg/mL) and ABTS method (47.2 ± 1.40, µg/mL) compared to standard ascorbic acid.


Introduction
Leguminosae is one of the most widespread plant families in the world, containing the genus Senna, which includes hundreds of plant species [1] such as Senna alexandrina, which is commonly known as Senna makkai or Cassia angustifolia. This species is native to Saudi Arabia, Egypt, and Yemen [2]. It is widely used in folk medicine for several purposes including as a purgative, antipyretic, laxative, and diuretic [3]. Several other proven and relevant biological activities such as anti-allergic, anti-inflammatory, antioxidant, antibacterial, antimicrobial, analgesic, antiparasitic, insecticidal, antitumor, hepatoprotective, and antifungal properties have been reported for several species of Senna [4]. The medicinal properties of Senna can be attributed mainly to the presence of anthraquinone glycosides, especially sennosides A and B [5], which act as pro-drugs for the laxative-acting anthrones and anthraquinones [6]. Sennosides A-D are mainly responsible for the purgative action of Senna [7], as intestinal bacteria metabolize sennosides to rheinanthrone, which acts as a direct purgative in the intestine [8]. Sennosides have been registered as one of the leading natural pharmaceutical preparations due to their important properties [9]. The content of sennosides A and B in raw materials determine their price and acceptability on the Several studies have previously been conducted for the extraction and quantification of active Senna constituents. However, the extraction optimization of important phytoconstituents sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol from Senna has not previously been undertaken. On this basis, we aimed to study the effects of different UAE extraction variables (extraction temperature, extraction time, and solid to liquid ratio) on the concentrations of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol using RSM. In addition, we also aimed to develop a validated HPLC method for the concurrent analysis of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol in optimized S. alexandrina samples.

Effect of Single-Factor Tests on Total Extraction Yield of S. alexandrina (Aerial Parts)
To set a range of UAE independent variables (extraction temperature, extraction time, and liquid to solid ratio) for optimization using the BBD method, we investigated the maximum single factor effects on the total percentage yield of S. alexandrina. Different ranges of extraction temperature (30-90 • C), extraction time (20-80 min), and solid to liquid ratio (8-32 mL/g) were selected to investigate the effect of individual independent factors (i.e., single factors) on total percentage yield while keeping the other two variables constant. Where these parameters were kept constant, an extraction temperature of 40 • C, extraction time of 30 min, and liquid to solid ratio of 20 mL/g were used. The results obtained showed that the total extraction yield was lowest at 30 • C extraction temperature, 20 min extraction time, and 8 mL/g liquid to solid ratio; the yields were highest at 70 • C, 40 min, and 24 mL/g, respectively. Setting the extraction temperature, extraction time, and liquid to solid ratio higher did not produce significant changes in yields ( Figure 2). The increase in temperature decreases the viscosity and surface tension of the solvent, increasing its matrix penetration power and resulting in enhanced extraction. Based on these observations, the independent extraction variable ranges were set as 50-70 • C extraction temperature, 30-60 min extraction time, and 16-32 mL/g of liquid to solid ratio; extraction optimization was then applied by the BBD method.

BBD Method Optimization of Extraction Conditions
The ranges for the three independent extraction variables, namely, extraction temperature (S 1 ), extraction time (S 2 ), and liquid to solid ratio (S 3 ), at three levels (+1, 0, −1) for extraction parameter optimization by the BBD method were selected based on observations from the single-factor experiments.

Statistical Analysis and Model Fitting
In Table 1, the results of 17 experimental combinations of three independent extraction variables were recorded to investigate their impact on percentage yields of sennoside A (R 1 ), sennoside B (R 2 ), aloe-emodin (R 3 ), emodin (R 4 ), and chrysophanol (R 5 ). The results were fitted into Equation (1) (i.e., a second-order polynomial equation):

BBD Method Optimization of Extraction Conditions
The ranges for the three independent extraction variables, namely, extraction temperature (S1), extraction time (S2), and liquid to solid ratio (S3), at three levels (+1, 0, −1) for extraction parameter optimization by the BBD method were selected based on observations from the single-factor experiments.
(where, Y = dependent variable (%yield); β 0 , β i , β ii , and β ij are the constant coefficient, linear coefficient of input factor P i , quadratic coefficient of input factor P i , and different interaction coefficients between input factor P i and P j , respectively;

BBD Method Optimization of Extraction Conditions
The ranges for the three independent extraction variables, namely, extraction temperature (S1), extraction time (S2), and liquid to solid ratio (S3), at three levels (+1, 0, −1) for extraction parameter optimization by the BBD method were selected based on observations from the single-factor experiments.
The adjusted R 2 /predicted R 2 values for R 1 , R 2, R 3 , R 4, and R 5 (0.9583/0.7795, 0.9656/0.8281, 0.9675/0.8462, 0.9760/0.8857, and 0.8169/0.7273, respectively) were found to be close to 1, showing a strong correlation between adjusted and predicted values. Furthermore, the difference between adjusted and predicted R 2 values was found to be less than 0.2 for every dependent variable, indicating that the models fitted well. To measure whether the precision is adequate, the signal-to-noise ratio can be used, which should be greater than 4 to fit the model. In this experiment, the signal to noise ratio for R 1 , R 2 , R 3 , R 4, and R 5 were 18.422, 20.067, 20.368, 23.618, and 8.202, respectively; all the values were more than 4, indicating that the models were fitted correctly and suggesting that the proposed model can be used to navigate the design space. In Table 3, the ANOVA (analysis of variance) results for the quadratic models of R 1 , R 2, R 3 , R 4 , and R 5 are listed. Table 1. Experimental parameters of Box-Behnken design and analysis of sennoside A (R 1 ), sennoside B (R 2 ), aloe-emodin (R 3 ), emodin (R 4 ), and chrysophanol (R 5 ) by HPLC-UV (n = 3).

Coded Variables
Sennoside A Yield (R 1 ) The model F-value for R 1 was found to be 41.81, implying that the model was significant and that there was only a 0.01% chance that an F-value this large could occur due to noise. Similarly, for R 2, R 3 , R 4 , and R 5 the F-values were found to be 50.97, 53.94, 73.19, and 8.93, respectively, implying that each quadratic model was significant and there was only a 0.01, 0.01, 0.01, and 0.43% chance, respectively, that these F-values could occur due to noise. The p-values for the proposed quadratic model of all the dependent variables were found to be very low (<0.05), suggesting that the models developed for analysis of all the variables were significant. The lack of fit F-values of 3.57, 2.89, 2.38, 2.42, and 0.20 for R 1 , R 2, R 3 , R 4, and R 5 imply that the lack of fit is not significant relative to the pure error; thus, it is appropriate to fit the model and predict the responses. There is a 12.52, 16.61, 21.04, 20.63, and 89.48% chance that a lack of fit F-value this large could occur due to noise for variables R 1 , R 2 , R 3 , R 4, and R 5 , respectively. The influences of individual independent variables S 1 , S 2 , and S 3 , as well as their various interactions with R 1 , R 2 , R 3 , R 4 , and R 5 , are listed in Table 4. The linear effects of two variables (S 1 , S 2 ) and quadratic effects of all three variables (S 12 , S 22 , and S 32 ) were found to be significant (p < 0.05), and affect the yields of R 1 , R 2 , R 3 , R 4 , and R 5 . The linear effect of the liquid to solid ratio (S 3 ) as well as the interaction effects of all three variables (S 1 S 2 , S 1 S 3 , and S 2 S 3 ) exhibited no significant effect (p > 0.05) on the yield of any of the dependent variables (R 1 , R 2 , R 3 , R 4 , and R 5 ). The high F-values of the linear and quadratic effects of S 1 indicated that, among all the dependent variables, S 1 has the most impact on the extraction dependent variables. Thus, the extraction will increase most with increasing extraction temperature but at high temperatures, the extraction will decrease. The R 2 /percentage coefficient of variation values for all dependent variables (R 1 , R 2 , R 3 , R 4 , and R 5 ) were 0.9817/2.06, 0.9850/1.96, 0.9858/1.85, 0.9895/1.53, and 0.9199/4.14, respectively, demonstrating good precision and reliability of the experimental values [24].
The response surface 3D plots and 2D contour plots (Figures 3-7) were designed to show the interaction effects of the independent variables (S 1 , S 2 , and S 3 ) on the yields of the dependent variables (R 1 , R 2 , R 3 , R 4 , and R 5 ). Each panel demonstrates the effect of two factors on the extraction yields while the third factor was fixed at a base level (60 • C for S 1 , 45 min for S 2 , and 24 mL/g for S 3 ). The effects of S 1 and S 2 (sub-figures A, B in Figures 3-7), S 1 and S 3 (sub-figures C, D in Figures 3-7), and S 2 and S 3 (sub-figures E, F in Figures 3-7) on R 1 , R 2 , R 3 , R 4 , and R 5 were recorded. Sub-figures A, B in Figures 3-7 showed that the extraction yields of the dependent variables were highest at S 1 of 63.8 • C and S 2 of 51.6 min. When S 1 exceeded 63.8 • C, the yield was found to decrease. The extraction yields of R 1 -R 5 increased when S 1 was set at 63.5 • C and S 3 at 25.6 mL/g, as shown in sub-figures C, D in Figures 3-7. Sub-figures E, F in Figures 3-7 demonstrate the effect of S 2 and S 3 interaction on the dependent variables; no significant changes were recorded with an increase in S 3 , however, a significant increase was observed with increasing S 2 . Based on these observations, we concluded that the maximal R 1 , R 2 , R 3 , R 4 , and R 5 could be extracted from the aerial parts of S. alexandrina using UAE at a temperature of 63.8 • C, extraction time of 51.6 min, and a liquid to solid ratio of 25.6 mL/g.

BBD Method Validation
The validation of the BBD method used for the analysis was performed by comparing the experimental values and predicted values of the responses (R 1 , R 2 , R 3 , R 4 , and R 5 ).
Establishing the suitability of the generated polynomial equation and BBD application was performed using the percentage prediction error, where small percentage prediction error values demonstrate the validity of the generated polynomial equation and application of the BBD model. The linear correlation between actual and predicted values of R 1 , R 2 , R 3 , R 4 , and R 5 demonstrated high R 2 values of 0.9923, 0.9949, 0.9918, 0.9972, and 0.9941, respectively, indicating excellent goodness of fit (p < 0.001).

Verification of Optimized Microwave-Assisted Extraction Conditions
The selected factors (S 1 -S 3 ) demonstrated diverse effects on the yield of dependent variables (R 1 -R 5 ) ( Table 5). The predicted optimal levels for R 1 -R 5 extraction were found to be S 1 of 63.8 • C, S 2 of 51.6 min, and S 3 of 25.6 mL/g, which were predicted to yield (% w/w) R 1 (2.152), R 2 (12.031), R 3 (2.331), R 4 (0.214), and R 5 (1.411). The predicted optimal level for each extraction factor was further modified to S 1 of 64.2 • C, S 2 of 52.1 min, and S 3 of 25.2 mL/g to obtain the maximum yields of R 1 -R 5 . The modified level of all extraction factors was used (n = 3) for the extraction of aerial parts of S. alexandrina using methanol (SAME) and the analysis of the obtained SAME was carried out using the developed HPLC-UV method to quantify R 1 -R 5 . By using the modified extraction condition, the amounts (% w/w) of R 1 , R 2 , R 3 , R 4 , and R 5 in SAME were found to be 2.237 ± 0.051%, 12.792 ± 0.475%, 2.457 ± 0.053%, 0.261 ± 0.0052%, and 1.529 ± 0.041%, respectively. The low residual percentages of 1.04, 1.06, 1.05, 1.22, and 1.08% for R 1 , R 2 , R 3 , R 4 , and R 5 , respectively, indicated that the model was reliable.

2,2-Diphenyl-1-Picrylhydrazyl (DPPH) and 2,2 -Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) Free Radical Scavenging Activities of SAME Samples
The DPPH radical scavenging activity for the 17 (S1-S17) SAME samples, along with the final BBD-optimized ultrasonic-extracted SAME, were evaluated, and the IC50 (µg/mL) values are shown in Table 6. Among these 17 SAME samples (S1-S17), many exhibited good antioxidant properties. Out of these 17 samples, S5 showed the maximum free radical scavenging property (IC50 = 68.4 ± 2.18 µg/mL) compared with standard ascorbic acid (IC50 = 5.9 ± 0.23 µg/mL). The SAME obtained after using the BBDoptimized ultrasonic extraction parameters exhibited strong antioxidant properties, having IC50 = 59.7 ± 1.93 µg/mL. Both the S5 and BBD-optimized SAME were found to contain a substantial amount of anthraquinone glycosides (sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol), as evaluated by using the HPLC-UV method, which may explain the samples' antioxidant properties. The results of ABTS radical scavenging activity of all SAME samples (S1-S17) as well as BBD-optimized ultrasonic-extracted samples given in Table 6 clearly indicate that the extracts (S5 and BBD-optimized SAME) containing larger amounts of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol (evaluated by HPLC-UV method) had greater free radical-scavenging properties (IC50 = 58.9 ± 2.33 and 47.2 ± 1.40 µg/mL, respectively). The optimization of extraction parameters leads to an increase in the extraction of all the standards (evaluated by HPLC analysis), which supported the increase in the free radical-scavenging property of the extract. Al-Zain et al. [25] found DPPH and ABTS antioxidant properties of methanol extract of S. alexandrina root with IC50 values of 235.9 and 321.5 µg/mL, respectively. Ahmed et al. [2] reported free radical-scavenging properties of methanol extract of C. angustifolia leaves with IC50 = 2.49 ± 0.01 µg/mL.

Discussion
In this experiment, we investigated the effects of various ultrasonic extraction parameters (extraction temperature, extraction time, and liquid to solid ratio) on sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol extraction from aerial parts of S. alexandrina using the BBD of RSM. The ultrasonication technique was previously reported to be the best method for sennoside A and sennoside B extraction from C. angustifolia leaves [26] as well as for the extraction of aloe-emodin, emodin, and chrysophanol from Rhamnus alpinus bark [27]. This was the basis of exploiting the ultrasonication extraction method and optimizing the extraction parameters by the BBD approach to obtain maximum yields from aerial parts of S. alexandrina. The BBD of RSM plays an important role in determining the optimal values of independent variables under which the maximum response of dependent variables can be achieved [28]. The response surface and contour plot approaches were used to visualize the correlation between responses and different levels of independent variables and interaction types between two independent variables [29]. In this experiment, at a fixed liquid to solid ratio and extraction time, the yields of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol increased with increasing extraction temperatures. Similar results were reported by Zhao et al. [30] and Ruan et al. [31], which may indicate that the solubility of these substances increases with increased temperature and, thus, the total yields also increase. Furthermore, the sennoside A, sennoside B, aloeemodin, emodin, and chrysophanol yields increased with increasing extraction time to a maximum, followed by a decline with further increases in extraction time. The decrease in extraction yields may be due to degradation of the phytoconstituents upon prolonged ultrasound action [32]. Mason et al. [33] reported that ultrasound may induce the formation of acoustic cavities, which prompted cracking of the plant cells. The plant cells cracked completely with increasing extraction time due to acoustic cavitation, thus increasing the extraction yields of the desired compounds. However, insoluble constituents, along with cytosol, become suspended in extraction solvent when the plant cells rupture, resulting in a reduction of the solvent permeability [34]. Additionally, the desired constituents become re-adsorbed on the ruptured plant particles because of their comparatively more specific surface areas, thus decreasing the yields [35]. A mathematical optimization was performed by using the BBD of RSM to decide the optimum level of different extraction variables to obtain maximum yields of the desired compounds. An extraction temperature of 63.8 • C, 51.6 min extraction time, and a 25.6 mL/g liquid to solid ratio were determined as the optimal extraction conditions for the ultrasonication technique. The maximum response values (% w/w) were predicted as 2.152, 12.031, 2.331, 0.214, and 1.411% of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol, respectively, under these operating conditions. The experiment was performed again with a modified extraction condition (extraction temperature of 64.2 • C, extraction time of 52.1 min, and 25.2 mL/g liquid to solid ratio) to obtain the maximum yield of the desired compounds. The extraction procedure with modified conditions was performed in triplicate, and the results of experimental values were compared with the predicted values. Mean values (% w/w) of 2.237% ± 0.051, 12.792% ± 0.475, 2.457% ± 0.053, 0.261% ± 0.0052, and 1.5297% ± 0.041, respectively, for sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol were obtained using the modified extraction procedure, which were very close to the predicted values, demonstrating the validity of the RSM model and also demonstrating that the suitability of the model for this extraction procedure.
Several phenolic substances, of both natural and synthetic origin, have been shown to act as antioxidant and radical scavengers, In particular, the flavonoids, which constitute an ubiquitous class as metabolites in higher plants, have been reported to contain numerous active substances [36,37]. In contrast, the anthraquinones and anthrones appear to be nearly unknown in this respect. However, it has been reported that substances of this class have interesting effects as antioxidants; some anthrones may have prooxidant activity due to their ability to generate superoxide anions [12]. It was found that anthraquinones such as chrysophanol and rhein accelerated the formation of hydroxyl radicals and among these compounds, emodin exhibited the strongest scavenging effect on hydroxyl radicals [38]. Emodin has been known to be pharmacologically potent and this characteristic was reported to be associated with its scavenging of hydroxyl radicals [39]. In contrast, the prooxidant ability of chrysophenol may derive from its greater formation of hydroxyl radicals [12]. In this experiment excellent antioxidant properties were exhibited by SAME obtained using the optimized extraction conditions with DPPH assay (IC50 = 59.7 ± 1.93, µg/mL) and ABTS methods (IC50 = 47.2 ± 1.40, µg/mL) compared to standard ascorbic acid. This supported the HPLC-UV analysis of antioxidant marker like emodin, chrysophenol in good quantity in SAME. There may be other compounds present in SAME along with these compounds that might support the antioxidant action of these markers synergistically.

Plant Material
The aerial parts of S. alexandrina (voucher specimen no. 16245) were collected in 2014 from Badr, Kingdom of Saudi Arabia by Dr. Md. Yusuf (field taxonomist, Pharmacognosy Department), and the specimen was deposited in the herbarium of Pharmacognosy Department, College of Pharmacy, King Saud University, Saudi Arabia. The collected aerial parts were cut into small pieces, washed with plain water, dried in the plant drying room in the Pharmacognosy Department, and stored in a tightly packed glass jar. Before extraction, the aerial parts were coarsely powdered using a grinder.

Ultrasound-Assisted Extraction of S. alexandrina Aerial Parts
The extraction of S. alexandrina powdered aerial parts (1 g) was executed in a 50 mL conical flask by UAE using a Sonics vibra cell (Model VCX-750; Sonics, Newtown, CT, USA) with methanol as solvent. Post-extraction, the SAME was cooled and filtered, and the residue was washed thrice with methanol to obtain the final SAME volume, which was then filtered using a syringe filter (0.45 µm, Phenomenex, Torrance, CA, USA). The final filtered extract was dried using a rotavapor (R-300, Buchi, Flawil, Switzerland) to obtain the final percentage yield.

Single Factor Experimental Design
The extraction variables (extraction temperature, time, and liquid to solid ratio) were selected based on observation of single-factor effects on the total extraction yield, which was used to optimize all extraction variables using the BBD method to obtain maximum sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol content from SAME. The impact of single factors on total extraction yield was evaluated by varying the extraction temperatures (30-90 • C), extraction times (20-80 min), and liquid to solid ratios (8-32 mL/g). When one factor was being used as a variable, the other two factors remained fixed (extraction temperature of 40 • C, extraction time of 30 min, and liquid to solid ratio of 20 mL/g).

Optimization of Extraction Variables Using BBD Method and Method Validity Testing
A 3-factorial (3 3 ) Box-Behnken design (version 13, Design-Expert Software, Stat-Ease Inc., Minneapolis, USA) was used for optimization of the independent variables extraction temperature (S 1 ), extraction time (S 2 ), and liquid to solid ratio (S 3 ), at low (−1), medium (0), and high levels (+1) ( Table 7). A total of 17 experimental runs, comprising five central points, were generated and fitted to a second-order polynomial equation to obtain the total yields of sennoside A (R 1 ), sennoside B (R 2 ), aloe-emodin (R 3 ), emodin (R 4 ), and chrysophanol (R 5 ). Two-dimensional contour plots and three-dimensional response surface plots were made to deduce the independent variables' effects on R 1 , R 2 , R 3 , R 4 , and R 5 yields, and the "biggest-is-best" principle was used for each variable to obtain the optimum outcome, with p-values ≤ 0.05 considered to be significant. A final confirmation experiment (n = 3) was performed using optimized independent extraction variables, and the experimental yields were compared with predicted values for model validation.

High-Performance Liquid Chromatography (HPLC) Analysis of BBD-run SAME Samples
For quantitative analysis of R 1 , R 2 , R 3 , R 4 , and R 5 in all 17 BBD-run SAMEs, as well as the final extract obtained using the optimized extraction conditions, an Alliance 2695 Separations module equipped with a 2487 dual wavelength absorbance detector (Waters Instruments, Inc., Milford, MA, USA), was used. The chromatographic analysis was performed using the following equipment: built-in quaternary pump, Pinnacle C18 column (5 µm, 250 × 4.6 mm), four-channel degasser, and autosampler with programmable temperature (25 • C). Different proportions of 0.5% formic acid in ultra-pure water (A) and acetonitrile (B) were used as a mobile phase with a flow rate adjusted to 1 mL/min.  The antioxidative properties of the 17 BBD-run SAMEs, as well as the final extract obtained using the optimized extraction conditions, were evaluated using DPPH, according to the earlier report by Alqahtani et al. [40], with minimal modifications. Different concentrations of the extracts (10, 50, 100, 500, and 1000 µg/mL) were prepared. Subsequently, 500 µL of each concentration was added to 125 µL of DPPH and 375 µL of methanol and then incubated for 30 min at room temperature. The antioxidant activity was measured spectrophotometrically (UV mini-1240, Shimadzu, Japan) in absorbance mode at λmax = 517 nm using ascorbic acid as the positive control. The following equation was used to calculate the radical-scavenging activity: % of radical-scavenging activity = (Abs control − Abs sample/Abs control) × 100

ABTS Radical Cation Scavenging Activity
The antioxidative assay of the 17 BBD-run SAMEs, as well as the final extract obtained by using the optimized extraction conditions, was evaluated by applying the ABTS method according to the previous report of Li et al. [41], with small modifications. Aqueous solutions of ABTS (7 mmol/L) and potassium persulfate (2.45 mmol/L) were prepared and, after 12 h in the dark, the two solutions were mixed and incubated for 0.5 h, kept in a refrigerator for 24 h, and then diluted in ethanol. To generate a calibration curve, a reaction was initiated by pipetting different prepared concentrations of each extract (10, 50, 100, 500, and 1000 µg/mL) to ABTS solutions of 50 µg/mL (1:1) and recording the absorbance at λmax = 734 nm using ethanol (95%), ABTS (50 µg/mL), and ascorbic acid as a blank, control, and standard, respectively. Three replicates for the standard and each extract were used for the analysis. The percentage of antioxidant property for each extract was calculated using the following formula [42]: % of radical scavenging activity = (Abs control − Abs sample/Abs control) × 100

Statistical Analysis
All experiments were carried out with three independent replicates and the values are presented as mean ± standard error of the mean (SEM). Data were statistically analyzed using the Student's t-test for comparison between the means, applying a significance level of p < 0.05.

Conclusions
In this study, RSM with a BBD was used to study ultrasound-assisted extraction of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol from the aerial parts of S. alexandrina. The experimental results demonstrated that all three extraction variables (extraction temperature, extraction time, and liquid to solid ratio), had an impact on the extraction of the target compounds. The optimal/modified extraction conditions were found to be: extraction temperature 63.8/64.2 • C, extraction time 51.6/52.1 min, and liquid to solid ratio 25.6/25.2 mL/g. Under these conditions, the experimental yields (% w/w) of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol were found to be 2.237, 12.792, 2.457, 0.261, and 1.529%, respectively, which agreed closely with predicted yields (2.152, 12.031, 2.331, 0.214, and 1.411%, respectively). The experimental values were consistent with values predicted by RSM models, thus demonstrating the fitness of the model employed and the accomplishment of RSM in optimizing the extraction conditions. An increase in the antioxidant property of the BBD optimized SAME against DPPH and ABTS radicals were supported by the increase in the content of antioxidant markers like emodin and chrysophenol (HPLC-UV anlysis). There may be some other antioxidant compounds present in BBD optimized SAME alongwith these markers which might synergistacally exhibited the excellent antioxidant propery of SAME. In the future, the proposed optimized ultrasonication extraction method in this work can be employed to extract sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol efficiently from marketed herbal supplements containing different Senna species.